Functional expression of secreted mouse BST-1 in yeast.
BST-1, a bone marrow stromal cell surface antigen, is a glycosyl phosphotidylinositol-anchored protein that stimulates pre-B-cell growth and has adenosine diphosphate (ADP)-ribosyl cyclase and cyclic ADP-ribose (cADPR) hydrolase activity. The two enzymatic activities are responsible for the synthesis and hydrolysis of cADPR, a novel second messenger of calcium release from intracellular calcium stores. The expression and characterization of human BST-1 in certain mammalian cell lines have been reported. We have expressed the murine BST-1 in yeast as a 6 x His-tagged secreted protein. The recombinant protein has been purified and subjected to structural and functional characterization. It has an apparent molecular mass of 38.5 kDa on SDS-PAGE gel stained with Coomassie blue and is recognized on Western blots by a rabbit polyclonal antibody against BST-1. Deglycosylation of the protein with N-glycanase produces a ladder of bands with molecular sizes ranging from 32 to 39 kDa. The protein possesses the ADP-ribosyl cyclase activity as measured using nicotinamide guanine dinucleotide as substrate.
Hussain
A M
AM
Department of Biochemistry, National University of Singapore, 119260, Singapore.
Lee
H C
HC
Chang
C F
CF
D036541:ADP-ribosyl Cyclase; D000818:Animals; D015703:Antigens, CD; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D058851:GPI-Linked Proteins; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007120:Immunochemistry; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics
D015703:Antigens, CD; D000954:Antigens, Surface; D058851:GPI-Linked Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D006639:Histidine; D036541:ADP-ribosyl Cyclase; C087521:ADP-ribosyl cyclase 2